V(D)J recombination is defective in human SCID, where RAG and Artemis mutations account for over one-third of human SCID patients. Knowledge of how the RAG proteins function will influence the choice of gene correction approaches in the cells from children with this form of SCID. Purified RAG proteins have been studied in their truncated forms (called core RAGs) since 1995, and this truncation removes what is called the non-core portion of RAG1 and 2. However, many RAG mutations in human SCID occur in the non-core portions. In the nearly-completed 4-year cycle of this grant, we made a major breakthrough in the V(D)J recombination field by generating the first completely full-length RAG protein complexes with consistently high enzymatic activity (Publ. 13 in the Progress Report publication list). We also demonstrated how RAG enzymatic behavior is stimulated by interaction with active histone tails (Publ. 7 in list). In the preliminary data for his project, we have developed a complete set of fluorescently tagged versions of our full-length RAG proteins and shown that these are still highly enzymatically active. This is a major advance for the RAG field because we can now follow full- length RAG action at the single molecule level for the first time using single-molecule Forster (fluorescence) resonance energy transfer (sm-FRET), as well as bulk solution (ensemble) FRET (en-FRET). This permits us to obtain clear data on many key steps in RAG function that are not obtainable using any other methods. First, what is the composition of the RAG complex during catalysis? RAG tetramers and octamers have both been proposed, and we bring sm-FRET, en-FRET and cryo-EM to bear on this question. Second, how does the RAG complex bring two recombination signal sequence (RSS) sites together (called synapsis)? This is fundamental, and yet we do not know whether one RAG complex must be bound at each RSS to achieve synapsis or whether one RAG:RSS complex can bind to a free partner RSS. Once formed, is this synaptic complex essentially irreversible? Third, how stably does the RAG complex hold the DNA ends, specifically the coding ends, after the double-strand breaks have occurred (in the post-cleavage complex, or PCC)? Does DNA sequence affect this stability? Do local histone tail modifications affect the PCC stability? Fourt, how does the RAG complex hand- off the coding ends to the NHEJ proteins? Must hairpin opening occur prior to this hand-off? Combining sm- FRET with other methods that we have undertaken, such as enzyme kinetics and cryo-EM, we are in a unique position to answer many central questions that have remained unclear in this field. Answers to these questions are central to understanding how mutations in the RAG proteins cause human SCID, and this knowledge can influence how gene correction or other therapies might best be developed for such patients in the future. Such information is also relevant in other areas, such as for T-cell lymphoma chromosomal translocations involving off-target cryptic RSS sites.